Regulation of folate receptor 1 gene expression in the visceral

Document Sample
Regulation of folate receptor 1 gene expression in the visceral Powered By Docstoc
					Ó 2009 Wiley-Liss, Inc.                                                              Birth Defects Research (Part A) 85:303À313 (2009)

        Regulation of Folate Receptor 1 Gene Expression
                    in the Visceral Endoderm
                            J. Michael Salbaum,1* Richard H. Finnell,2 and Claudia Kappen1
                          Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana
                  Institute for Biosciences and Technology, Center for Environmental and Genetic Medicine, Houston, Texas
                              Received 31 July 2008; Revised 7 September 2008; Accepted 14 September 2008

BACKGROUND: Nutrient supply to the developing mammalian embryo is a fundamental requirement.
Before completion of the chorioallantoic placenta, the visceral endoderm plays a crucial role in nurturing the
embryo. We have found that visceral endoderm cells express folate receptor 1, a high-affinity receptor for
the essential micronutrient folic acid, suggesting that the visceral endoderm has an important function for
folate transport to the embryo. The mechanisms that direct expression of FOLR1 in the visceral endoderm
are unknown. METHODS: Sequences were tested for transcriptional activation capabilities in the visceral
endoderm utilizing reporter gene assays in a cell model for extraembryonic endoderm in vitro, and in trans-
genic mice in vivo. RESULTS: With F9 embryo carcinoma cells as a model for extraembryonic endoderm, we
demonstrate that the P4 promoter of the human FOLR1 gene is active during differentiation of the cells
towards visceral endoderm. However, transgenic mouse experiments show that promoter sequences alone
are insufficient to elicit reporter gene transcription in vivo. Using sequence conservation as guide to choose
genomic sequences from the human FOLR1 gene locus, we demonstrate that the sequence termed F1CE2
exhibits specific enhancer activity in F9 cells in vitro, in the visceral endoderm, and later the yolk sac in
transgenic mouse embryos in vivo. We further show that the transcription factor HNF4-alpha can activate
this enhancer sequence. CONCLUSIONS: We have identified a transcriptional enhancer sequence from the
FOLR1 locus with specific activity in vitro and in vivo, and suggest that FOLR1 is a target for regulation by
HNF4-alpha. Birth Defects Research (Part A) 85:303–313, 2009. Ó 2009 Wiley-Liss, Inc.

Key words: folate receptor; transcriptional regulation; visceral endoderm; enhancer; HNF4-alpha

                 INTRODUCTION                                           Shaw et al., 1995), represented a significant milestone in
     Folate, Birth Defects, and General Health                          the understanding of congenital malformations. Impor-
                                                                        tantly, this concept presented a highly feasible therapeu-
   Folate deficiency has been linked to an increased inci-               tic approach to birth defect prevention simply by supply-
dence of congenital malformation (Molloy and Scott,                     ing vitamin preparations including folate to women who
2001), heightened risk for certain types of cancer (Prinz-              wished to become pregnant (Smithells et al., 1981). In
Langenohl et al., 2001; Ryan and Weir, 2001; Courte-                    fact, periconceptional supplementation with folate (Lock-
manche et al., 2004b), reduced immune system perform-                   smith and Duff, 1998; Bailey, 2000; Ladipo, 2000) proved
ance (Courtemanche et al., 2004a), anemia, and reduced                  to be highly beneficial to the conceptus, resulting in sig-
endurance (Lukaski, 2004). Suboptimal folate levels                     nificantly decreased occurrence of NTDs, craniofacial
appear to be linked to impaired general health (Singh,                  malformations, and cardiovascular abnormalities among
2004) and neurologic symptoms in aging (D’Anci and                      newborns (Wald et al., 1991; Gelineau-van Waes and Fin-
Rosenberg, 2004; Kim et al., 2008). Furthermore, our own                nell, 2001).
experiments have demonstrated a beneficial effect of fo-
late on the morphogenesis of the skeleton (Kappen et al.,
2004). Historically, the finding that neural tube defect                 Presented at the 48th Annual Meeting of the Teratology Society, June 28–July
(NTD) frequencies may be associated with low folate lev-                2, 2008, Monterey, CA.
els in the mother (Smithells et al., 1976; Yates et al., 1987;          *Correspondence to: J. Michael Salbaum, 6400 Perkins Road, Baton Rouge,
                                                                        LA 70808. E-mail:
Milunsky et al., 1989), and the resulting general hypothe-              Published online 29 January 2009 in Wiley InterScience (www.interscience.
sis that nutritional deficiencies could be involved in the     
etiology of birth defects (Smithells et al., 1976, 1977;                DOI: 10.1002/bdra.20537

                                              Birth Defects Research (Part A): Clinical and Molecular Teratology 85:303À313 (2009)
304                                                   SALBAUM ET AL.
         Folate Transport and Cellular Uptake                   appear that the FOLR1 protein represents the gateway
                                                                for this important micronutrient through the visceral
   Mammalian cells have developed an elaborate mecha-
                                                                endoderm to the embryo itself. It is therefore possible
nism to harvest extracellular folate (Trippett and Bertino,
                                                                that the lack of Folr1 in the gene knockout model may
1999), involving extracellular, glycolipid-anchored high-
                                                                not only have a cell-autonomous effect on cells of the
affinity folate receptors (Lacey et al., 1989; Wang et al.,
                                                                neural tube, but an additional indirect, pleiotropic effect
1996; Wu et al., 1997), a low-affinity transmembrane car-
                                                                on the whole embryo. Such pleiotropy may arise from
rier (Moscow et al., 1995; Wong et al., 1995), and a pro-
                                                                the absence of Folr1 in the visceral endoderm, a resulting
ton-coupled folate transporter (Qiu et al., 2006). Five fo-
                                                                defect in folate transport in the visceral endoderm, failure
late receptor genes have been reported for the human ge-
                                                                to supply folate from the visceral endoderm to the
nome (Elwood, 1989; Lacey et al., 1989; Ross et al., 1994;
                                                                embryo, and consequently, a condition of folate defi-
Spiegelstein et al., 2000), whereas in the mouse, three fo-
                                                                ciency throughout the embryo, with negative consequen-
late receptor genes are present. Expression studies on
                                                                ces for cell proliferation and normal morphogenesis.
human folate receptors reveal that FOLR1 is mostly
expressed in epithelial cells (Lacey et al., 1989; Page
et al., 1993; Smith et al., 1999), FOLR3 is specific for the             Functions of the Visceral Endoderm
hematopoietic system (Shen et al., 1994), and FOLR2                The visceral endoderm has important roles in pattern-
(Reddy et al., 1999; Ross et al., 1999; Shen et al., 1994)      ing and in nurturing the developing embryo (Brent et al.,
and FOLR4 (Spiegelstein et al., 2000) are found at lower        1990; Bielinska et al., 1999). Crucial patterning signals for
levels in diverse tissues. FOLR4 may play a role for the        determination of anterior identity arise from the anterior
immune system because of its expression on regulatory           visceral endoderm (Thomas and Beddington, 1996; Tam
T-cells (Walker, 2007; Yamaguchi et al., 2007). The carrier     and Behringer, 1997; Beddington and Robertson, 1998),
protein encoded by the RFC1 gene and the proton-                whereas visceral endoderm adjacent to extraembryonic
coupled folate transporter PCFT seem to be widely dis-          mesoderm is essential for the induction of blood vessel
tributed (Said et al., 1996; Wang et al., 2001; Maddox          development (Boucher and Pedersen, 1996; Belaoussoff
et al., 2003; Qiu et al., 2007). Mouse embryos lacking the      et al., 1998). In parallel, the visceral endoderm is respon-
Folr1 (folbp1) gene (Piedrahita et al., 1999) are arrested in   sible for nutrient uptake and transport to the embryo
their development shortly after gastrulation, fail to close     (Cross et al., 1994). This function for nutritional support
the neural tube, and die in utero at mid-gestation, dem-        is indispensable during establishment of the chorioallan-
onstrating that the mouse Folr1 gene is essential for em-       toic placenta (Brent et al., 1990); once the placenta is
bryonic development.                                            functional, the embryo can switch from histiotrophic to
                                                                hemotrophic nutrition (Burton et al., 2001). However, it is
                                                                important to consider that crucial developmental proc-
      Expression of Folate Receptor Genes during                esses, such as neural tube closure and patterning of the
        Embryonic Development in the Mouse                      early heart, occur at a time when the burden of nurturing
                                                                the embryo lies with the visceral endoderm (Brent et al.,
   The essential nature of folate intuitively would suggest
                                                                1990). Thus, it stands to reason that birth defects involv-
that genes involved in folate transport and processing
                                                                ing the early embryonic patterning processes in relation
would be ‘housekeeping’ genes, with expression in every
                                                                to a lack of nutrients should be interpreted in the context
cell. In contrast, published observations (Saitsu et al.,
                                                                of visceral endoderm function. Examples are spina bifida
2003) and our own in situ hybridization experiments
                                                                and folate deficiency (Smithells et al., 1976), or diabetic
(Kappen et al., 2004) demonstrate that this is clearly not
                                                                embryopathy and the detrimental nutritional milieu
the case: in the mouse embryo, genes for folate receptors
                                                                brought about by maternal diabetes (Reece et al., 1993;
are expressed in distinct and specific tissue distributions
                                                                Reece and Eriksson, 1996). In fact, gene expression
during development. The expression of Folr1 in the neu-
                                                                changes in the visceral yolk sac are thought to contribute
ral tube appears to represent a direct link to NTDs
                                                                to birth defects in diabetic pregnancies (Reece et al.,
through a cell-autonomous function of the Folr1 gene
                                                                2006). Therefore, proper regulation of genes involved in
(Saitsu et al., 2003), and folate supplementation is able to
                                                                nutrient uptake and transport in the visceral endoderm is
rescue embryos lacking the Folr1 gene (Spiegelstein et al.,
                                                                a crucial prerequisite for successful development of the
2004). However, the literature is not clear about the
                                                                embryo itself. A targeted mutation of the transcription
causes for neurulation defects: both neural tube cells
                                                                factor HNF4-alpha underscores that view, as many genes
(Copp, 2005) and cells adjacent to the neural tube (Copp
                                                                for nutrient transport or metabolism, such as apolipopro-
et al., 1988; van Straaten et al., 1993) are being implicated
                                                                teins, glucose transporter 2, transferrin, and cytoplasmic
in a role for NTDs. Thus, a cell-autonomous model for
                                                                retinoic acid binding proteins are downregulated in the
Folr1 in NTDs may explain only part of the involvement
                                                                visceral endoderm of HNF4-alpha2/2 embryos (Stoffel
of folate in neural tube closure.
                                                                and Duncan, 1997). In fact, the failure of HNF4-alpha2/2
   In this context, it was interesting to note that the ear-
                                                                embryos to complete gastrulation has been ascribed to
liest and strongest expression signal for Folr1 in the
                                                                ‘‘death by starvation’’ (Copp, 1995; Duncan et al., 1997).
developing embryo occurred in cells of the visceral endo-
derm (Saitsu et al., 2003), and our own data (Fig. 1).
Expression was conspicuously absent from the embryo             Regulation of Folate Receptor 1 Gene Expression
itself, raising the question as to how folate enters the ma-      Gene transcription is typically dependent on regulatory
jority of cells in the embryo during crucial periods of         DNA elements such as promoters and enhancers. Like all
morphogenesis. Regarding the visceral endoderm, we              folate receptor genes, the human FOLR1 gene has multi-
found that Folr1 was the only gene of the folate receptor       ple promoters that are well characterized. The FOLR1 P1
family expressed in this tissue. Consequently, it would         promoter (so designated as the transcript starts at exon 1)

Birth Defects Research (Part A) 85:303À313 (2009)
                           TRANSCRIPTIONAL ENHANCER FOR FOLATE RECEPTOR 1                                             305
was studied in KB epidermal carcinoma cells and NIH/                     MATERIALS AND METHODS
3T3 fibroblasts (Elwood et al., 1997; Galmozzi et al.,                      In Situ Hybridizations
2001). Both the FOLR1 P1 and the FOLR1 P4 promoter
(transcript starting at exon 4) contain initiator sequen-         Decidua with mouse embryos at 7.5 days’ gestation—
ces and respond to the transcription factor SP1 (Sada-         with 12 PM on the day of the appearance of a vaginal
sivan et al., 1994; Saikawa et al., 1995). The two pro-        plug designated as gestation day 0.5—were dissected
moters exhibit differential activity in KB cells, several      from the uterus of FVB mice, embedded in O.C.T. com-
adult tissues, and ovarian cancer cells (Elwood et al.,        pound (Sakura Finetek, Torrance, CA), frozen, and used
1997; Galmozzi et al., 2001). Furthermore, the transcrip-      to generate cryosections of 25 lm thickness. A digoxige-
tion factor vHNF1 activates the P1 promoter in ovarian         nin-labeled antisense riboprobe was generated from a
carcinoma cells (Tomassetti et al., 2003), and the FOLR1       mouse Folr1 full-length cDNA clone, and sections were
P4 promoter is modulated by the estrogen receptor in           hybridized as described previously (Salbaum, 1998).
cervical and ovarian carcinoma cells (Kelley et al.,
2003). Interestingly, in several cell lines, the FOLR1                          Plasmid Constructs
gene appears to regulated in response to cellular                Reporter constructs for Luciferase assays were gener-
growth and not in response to folate levels (Doucette          ated in pGL3 (Promega, Madison, WI). DNA fragments
and Stevens, 2001), indicating that cellular requirements      spanning the P1 and P4 promoter regions, as well as evo-
rather than a simple feedback mechanism control this           lutionary conserved sequences flanking the human
gene. A recent study reported genetic variation in the         FOLR1 gene, were generated by PCR from commercially
FOLR1 promoter region (Nilsson and Borjel, 2004), with         obtained human genomic DNA (Roche, Indianapolis, IN).
potential health implications presumed to be due to            Genomic coordinates (UCSC genome browser, human ge-
altered expression of the gene. As is evident from the         nome version hg18, March 2006 assembly) for the ampli-
literature on folate receptor gene promoters, most             fied fragments were as follows: promoter P1, chr11:
experiments have focused on carcinoma cell lines, with         71,576,404-71,578,395; promoter P4, chr11:71,578,925-
emphasis on FOLR1 gene regulation in cancer.                   71,580,883. For conserved sequence elements from the
   In contrast, regulatory mechanisms for FOLR1 gene           FOLR1 gene, we use the abbreviation F1CE (for Folate re-
expression during embryonic development have not been          ceptor 1 Conserved Element) followed by a number;
explored. The expression of FOLR1 in the visceral endo-        genomic coordinates were: F1CE1, chr11:71,560,901-
derm is consistent with a role of the visceral endoderm        71,561,809; F1CE2, chr11:71,565,324-71,566,907; F1CE3,
in folate uptake and subsequent release to cells of the        chr11:71,591,596-71,592,126. The identity of each ampli-
embryo itself. Therefore, the mechanism that is responsi-      fied DNA fragment was confirmed by DNA sequencing.
ble for the specific expression of Folr1 in the visceral        Promoter fragments were generated with flanking MluI
endoderm is fundamental to ensure folate supply to the         and XhoI restriction sites for cloning into pGL3
embryo. To identify this mechanism, we have undertaken         (Promega); fragments carrying conserved sequences were
reporter gene experiments to gain further insight into the     produced with flanking KpnI and MluI sites for cloning
regulatory events that control Folr1 expression during de-     upstream of promoter sequences. Deletions in F1CE2-
velopment, with our focus on the visceral endoderm.            F1P4-GL3 were generated using existing restriction
   Given the importance of folate for prevention of            enzyme sites. A reporter plasmid carrying HcRed as re-
human birth defects, it is reasonable to assume that defi-      porter gene was generated by replacing the coding
ciencies in folate transport may be cause for susceptibility   sequence for luciferase in pGL3 with the HcRed coding
to congenital malformation in humans. Such deficiencies         sequence from pHcRed-N1.1 (Clontech, Mountain View,
might arise from genetic variation in the structural part      CA). The plasmid F1CE2-F1P4-GhcR contains the same
of the FOLR1 gene, but could also be based on mutations        assembly of conserved sequence and promoter as F1CE2-
in regulatory regions that are required for proper expres-     F1P4-GL3 in the context of the fluorescent reporter, and
sion of FOLR1. Identifying the human regulatory ele-           was used for transgenic mouse experiments.
ments for FOLR1 expression would provide a means to
characterize genetic variation in such elements, and
investigate the relationship to birth defect susceptibility.                 Transfection Experiments
To date, this approach was limited to promoter regions            Conditions to grow F9 mouse embryo carcinoma cells
of FOLR1 (Barber et al., 2000), because the existence,         as well as differentiation to either visceral or parietal
identity, and location of enhancer sequences were              endoderm phenotype were as described elsewhere
unknown. Therefore, rather than using murine sequen-           (Braunhut et al., 1992; Dong et al., 1990). Cells were
ces, we chose to attempt identification of regulatory           seeded in 35-mm dishes at a density of 1 to 2 3 105 cells
sequences from the human FOLR1 locus, using a trans-           per dish; transfections using Effectene (Qiagen, Valencia,
genic mouse approach to provide an evolutionary con-           CA) and 400 to 600 ng of plasmid DNA were performed
served in vivo context. In this fashion, any human             24 hours after plating. Twenty-four hours after transfec-
sequences with regulatory function in vivo may be read-        tion, the transfection mixture was replaced with media
ily checked for potential genetic variation in human pop-      containing differentiation agents; for visceral endoderm,
ulations in the future. In this study, we report the identi-   cells were treated with 1 lM all-trans retinoic acid
fication of a sequence from the human FOLR1 locus that          (Sigma, St. Louis, MO), whereas for parietal endoderm,
can act as a transcriptional enhancer to direct gene           cells were grown on dishes pretreated with 0.1% gelatin
expression specifically in the visceral endoderm, and we        and received 1 lM all-trans retinoic acid and 250 lM
suggest that the FOLR1 gene, like many other genes for         dibutyryl-cAMP (Sigma). Sixty hours after induction of
nutrient uptake or transport, is a target for the transcrip-   differentiation, a time at which any retinoic acid-medi-
tion factor HNF4-alpha.                                        ated effects of FOLR1 have long ceased, cells were lysed

                                                                         Birth Defects Research (Part A) 85:303À313 (2009)
306                                                  SALBAUM ET AL.
in GloLysis buffer (Promega), and luciferase activity was
determined using SteadyGlo substrate (Promega). Lucif-
erase values were normalized to the protein content of
the lysate as determined by BCA assay. Each transfection
assay was performed in either five or ten replicates. For
cotransfections, expression vectors encoding transcription
factors (CMV-HNF4-alpha, CMV-TGIF) were obtained
from the IMAGE Mammalian Gene Collection of full-
length cDNAs (Open Biosystems, Huntsville, AL). The
expression vector pMT7-HNF4-alpha (Jiang et al., 1995)
was kindly provided by Dr. Francis Sladek (University of
California, Irvine, CA). DNA for cotransfection experi-
ments was a mixture of 500 ng of reporter construct
DNA as well as 100 ng DNA of the plasmid encoding a
transcription factor. An expression vector containing the
HcRed fluorescent protein sequence was used as negative
control (in place for transcription factor-expressing plas-    Figure 1. Expression of the mouse Folr1 gene in the visceral
mids) for cotransfection experiments and served for nor-       endoderm. Sagittal (A) and transverse (B) sections from mouse
malization of reporter activity. For all experiments, fold     decidua at 7.5 days’ gestation were hybridized with an antisense
changes were calculated by normalizing all observed val-       riboprobe specific for the murine Folr1 gene. Strong expression
ues to the average of the respective control experiment.       was observed in the visceral endoderm, with weaker signal in
Statistical significance was determined by performing a         the chorion, and no expression detectable in the embryo itself.
double-sided t test on control and experimental values         Abbreviations: ave, anterior visceral endoderm; ch, chorion; d,
normalized to the average of the controls.                     deciduum; ec, ectoderm; h, headfold region; m, mesoderm; ve,
                                                               visceral endoderm.

           Transgenic Mouse Experiments
   The construct F1CE2-F1P4-GhcR was used to generate            Activity of FOLR1 Gene Promoters in F9 Cells
transgenic mouse embryos that were then analyzed for               Differentiated toward Visceral Endoderm
reporter activity by confocal microscopy. Injection DNA
free of plasmid backbone sequences was generated by               We generated reporter constructs comprising 2 kb of
digestion with Asp718 and SalI, followed by agarose gel        sequences of either the P1 or the P4 promoter of the
electrophoresis and purification (Qiagen). DNA was              human FOLR1 gene (Fig. 2A) to test their activity in F9
injected into fertilized oocytes of FVB mice as published      embryo carcinoma cells that were differentiated to vis-
(Hogan et al., 1996). At gestation day 7.5 (E7.5) as well as   ceral endoderm (Dong et al., 1990; Braunhut et al., 1992).
9.5 (E9.5), embryos were dissected from the uterus of          Although we observed no activity from the P1 promoter
CD-1 foster mice, and embryo as well as yolk sac (at           in F9 cells under any circumstance, the construct carrying
E9.5) of each specimen were used for imaging HcRed-            the P4 promoter showed activity in F9 cells, but only af-
specific fluorescence on a Zeiss Confocal microscope             ter they had undergone differentiation toward a visceral
(Carl Zeiss Inc., Thornwood, NY); all images were taken        endoderm phenotype (Fig. 2B). In F9 cells grown without
at identical intensity settings. Genotyping for transgene      induction of differentiation, the P4 construct did not ex-
presence was performed on DNA extracted from                   hibit any activity higher than a promoterless luciferase
embryos after imaging.                                         control vector. We conclude that the P4 promoter of the
                                                               human FOLR1 gene has the potential to contribute to
                                                               expression of the gene in the visceral endoderm.
  Expression of Folr1 in the Visceral Endoderm                          Activity of FOLR1 Gene Promoters
   To reveal sites of expression of the Folr1 gene, we per-                     in Transgenic Mice
formed in situ hybridization experiments on mouse                 With the observation of cell type-specific promoter ac-
embryos at stages prior to neural tube closure. While our      tivity from the P4 promoter in the visceral endoderm
results in general confirm previously published data            model, we introduced reporter constructs with a b-galac-
(Saitsu et al., 2003), we were intrigued by the high level     tosidase reporter gene (Fig. 2A) in transgenic mouse
of expression of Folr1 in the visceral endoderm (Fig. 1) at    embryos to test whether promoter sequences of the
embryonic day 7.5, and the yolk sac at later stages of de-     human FOLR1, or the mouse Folr1 gene, were sufficient
velopment. The visceral endoderm is a cell layer thought       to drive expression of a reporter gene in a pattern resem-
to play an important role for nutrition of the embryo          bling the expression of Folr1 in the mouse. Although we
(Brent et al., 1990). We detected only Folr1 expression;       were able to generate transgenic specimen at expected
neither Folr2 nor Folr4 expression was found (not shown).      frequencies (Table 1), none of those transgenic specimen
It is therefore likely that Folr1 represents the gateway for   showed reporter activity that matched expression of
high-affinity folate transport in the visceral endoderm,        Folr1. A few embryos transgenic for the human FOLR1
and the regulatory mechanisms that direct expression of        P4 construct displayed some lacZ activity, but the spatial
the Folr1 gene in the visceral endoderm are likely to be       distributions of these activities were not consistent
of high biologic significance for healthy development of        between individual transgenic samples, and did not
the embryo.                                                    match the known Folr1 expression pattern (Saitsu et al.,

Birth Defects Research (Part A) 85:303À313 (2009)
                              TRANSCRIPTIONAL ENHANCER FOR FOLATE RECEPTOR 1                                                           307

Figure 2. Activity of the P4 promoter of the human FOLR1 gene. (A) Reporter constructs from the human FOLR1 gene. Promoter con-
structs included the publicly annotated transcription start site as well as 2 kb of upstream DNA for each respective construct. Firefly Lu-
ciferase as well as Escherichia coli b-galactosidase were used as reporter genes. (B) Reporter construct from the mouse Folr1 gene locus.
(C) The human P4 promoter construct shows specific activity in F9 embryo carcinoma cells only after differentiation towards visceral

2003). We did not observe any reporter activity from                    sequence F1CE2 to the P4 promoter resulted in an
transgenic samples carrying either the mouse Folr1 P1 or                approximately eightfold increase of reporter activity in
the mouse Folr1 P4 construct. We therefore conclude that                the visceral endoderm differentiation model, suggesting
the individual P1 or P4 promoter sequences of either the                that the F1CE2 sequence can in fact act as an enhancer.
human or the mouse folate receptor 1 gene are not suffi-                 Deletion of approximately two thirds of the F1CE2
cient to drive gene expression in the correct pattern                   sequence between the Asp718 and StuI restriction sites
in vivo.                                                                (F1CE2-dAS-F1P4) abolished that enhancement, suggest-
                                                                        ing that this enhancing activity may reside between these
   Conserved Sequence Elements at the FOLR1                             two coordinates. Deletion of the sequence between the
                                                                        two PflI restriction sites had little effect on enhancer ac-
                 Gene Locus                                             tivity, as seen for the F1CE2-dP-F1P4 and F1CE2-dPSM-
   Because the gene for folate receptor 1 showed a high                 F1P4 constructs. This suggests that the sequence between
degree of sequence conservation between human and                       the second PflI site and the StuI site, which also contains
mouse, we hypothesized that the regulatory mechanisms                   a conserved sequence element, is a major contributor to
controlling the expression of the gene might also be con-               the observed enhancer effect.
served. We used VISTA to generate a sequence conserva-
tion landscape around the human FOLR1 gene (Fig. 3).
                                                                         Tissue-Specific Enhancer Activity of a Conserved
We initially selected the three conserved regions nearest
to the FOLR1 gene and generated reporter constructs                          Sequence Upstream of the FOLR1 Gene
where each of the conserved sequences were placed in                       To determine whether the F1CE2 sequence would be
the context of the FOLR1 P4 promoter. An initial transfec-              able to confer enhancer activity in vivo, we generated
tion survey experiment with constructs containing a fluo-                transgenic mice with a construct carrying the same
rescent reporter (HcRed) suggested that the sequence                    F1CE2-F1P4 configuration as described in the in vitro
termed F1CE2 conferred transcriptional activation activity              experiment, but using a gene for the red fluorescent pro-
upon the P4 promoter. We therefore decided to examine                   tein HcRed as the reporter gene. We performed transient
the F1CE2 sequence in further detail.                                   transgenic assays where the analysis for reporter activity
                                                                        was carried out directly on founder embryos. Analysis of
 A Conserved Sequence Upstream of the FOLR1                             embryos at 7.5 days of gestation (Fig. 5) revealed that all
                                                                        eight transgenic embryos exhibited red fluorescence re-
    Gene Shows Enhancer Activity in vitro
                                                                        stricted to the region of the visceral endoderm. A second
  Using firefly luciferase as the reporter gene, we com-                  experiment analyzed at 9.5 days of gestation yielded
pared reporter activity for DNA constructs containing the               three transgenic specimen; all three showed consistent
FOLR1 P4 promoter in the presence or absence of the                     red fluorescence in the yolk sac. Fluorescence signals in
F1CE2 sequence, or with parts of the F1CE2 sequence                     embryos were spurious or not detectable at all, indicating
deleted from the construct. We tested these constructs in               that the reporter activity from this construct was specific
F9 cells differentiated either towards the visceral or the
parietal endoderm model. The results are summarized in
Figure 4. Compared to the promoter-less vector pGL3,                                            Table 1
presence of the P4 promoter resulted in an increase in re-                       Reporter Constructs in Transgenic Mice
porter activity at about the same magnitude as observed                 Construct           Embryos         Transgenic         Expression
in the initial experiment; this was observed for both cell
models. Activity of the F1P4-GL3 construct was then                     hF1P4-LacZ            125               18                 0
used to normalize reporter activities and calculate fold                mf1P4-LacZ            106               16                 0
                                                                        mf1P1-LacZ            130               10                 0
change. We found that addition of the conserved

                                                                                    Birth Defects Research (Part A) 85:303À313 (2009)
308                                                      SALBAUM ET AL.

Figure 3. Conservation profile at the human FOLR1 gene locus. Sequence conservation plot in the vicinity of the human FOLR1 gene
locus. Genes are annotated by arrows, conserved sequence regions used in this study are outlined. Annotation of conservation peaks fol-
lows the VISTA convention, with conserved coding regions colored purple, transcribed non-coding regions in light blue, and conserved
noncoding regions in pink. The colored bar above the conservation landscape indicates the presence of repetitive elements in the human
sequence. Three sequences (termed F1CE, for FOLR1 Conserved Element) with conservation to multiple species were initially chosen to
be included in reporter constructs and tested for transcriptional activation. Both F1CE1 and F1CE2 show deep conservation across verte-
brates, whereas conservation in the F1CE3 sequence is limited to mammals.

for the visceral endoderm at E7.5, and for the yolk sac at
E9.5, but not for the embryo proper. This was in excellent
agreement with our earlier in situ hybridization results
on the expression of the Folr1 gene itself, and suggests
that the human F1CE2 region contains an enhancer
sequence that is sufficient to drive the reporter gene
expression in vivo in a pattern that resembles the expres-
sion of the mouse Folr1 gene. Based on the consistency
between the in vitro and the in vivo results, we conclude
that the F1CE2 sequence can function as an enhancer in
the regulation of folate receptor gene expression in early
                                                                      Figure 4. Enhancer activity from a DNA fragment upstream of
 HNF4-Alpha Can Activate the FOLR1 Enhancer                           the human FOLR1 gene. F9 embryo carcinoma cells were trans-
                                                                      fected with various DNA constructs and differentiated either
   When we examined the PflI-StuI fragment of the                      towards visceral or towards parietal endoderm. From top: pGL3,
F1CE2 region for conservation and for the presence of                 basic Luciferase vector without promoter sequences; F1P4-GL3,
potential transcription factor binding sites, we noted a              human FOLR1 P4 promoter construct as reference for the experi-
sequence 50 -TGGAATTGGACCT-30 that was identified by                   ment; F1CE2-F1P4-GL3, conserved sequence F1CE2 tagged onto
rVISTA software (Loots et al., 2002; Loots and Ovchar-                the human FOLR1 P4 promoter construct; F1CE2-dP-F1P4, dele-
                                                                      tion in the conserved F1CE2 sequence between the two PflI
enko, 2004) as a potential binding site for the transcrip-            restriction sites; F1CE2-dAS-F1P4, deletion between Asp178 and
tion factor HNF4-alpha. This suggested the possibility                StuI sites; F1CE2-dSM-F1P4, deletion between StuI and MluI
that HNF4-alpha might be involved in the function of the              sites; F1CE2-dPSM-F1P4, compound deletion with the sequence
F1CE2 enhancer function and thereby contribute to the                 between the PflI sites as well as the sequence between the StuI
regulation of the folate receptor 1 gene. We tested this              and MluI sites absent from the F1CE2 sequence. All deletion
possibility by performing cotransfection experiments in               constructs share the F1P4-Luciferase portion. Restriction sites
F9 cells differentiated towards the visceral endoderm. For            and conservation regions (black) in the F1CE2 sequence are indi-
these experiments, we compared the reporter activity of               cated to the right; gray bars represent the sequence present in
                                                                      the various deletion constructs. The presence of the F1CE2
the F1P4 promoter alone, the F1P4 promoter carrying the
                                                                      sequence enhances the P4 promoter activity nearly eightfold in
full sequence of the F1CE2 region, and the F1P4 pro-                  the visceral endoderm paradigm. F1P4 constructs also show ac-
moter with the F1CE2-dPSM deletion. Luciferase reporter               tivity in the parietal endoderm model, although the enhancing
plasmids were cotransfected with expression vectors that              function of the F1CE2 sequence is diminished. Deletion of the
would express (1) HNF4-alpha from the MT7 promoter                    sequence between the Asp718 and StuI sites from the F1CE2
(Jiang et al., 1995), (2) HNF4-alpha from the CMV pro-                sequence abolished the enhancement of transcription activity.

Birth Defects Research (Part A) 85:303À313 (2009)
                              TRANSCRIPTIONAL ENHANCER FOR FOLATE RECEPTOR 1                                                         309

Figure 5. Enhancer activity of the F1CE2 sequence in transgenic mice. (A, B, C, D) Images (confocal slices) of four different embryos at
E7.5 that all carry the F1CE2-F1P4-HcRed transgene. Red fluorescence appeared to be restricted to the layer of visceral endoderm (ve)
cells on the outside of the embryo. Stippled white lines show the location of the embryo (e) and the chorion (ch); stippled yellow lines
mark the approximate boundary of the visceral endoderm (ve). (E) Yolk sac (ys) from a transgenic specimen at E9.5 showing bright red
fluorescence (projection view of a stack of confocal images), which is indicative of high reporter activity. (F) Transgenic embryo (slice
view) corresponding to the yolk sac shown in E with very little reporter fluorescence. Stippled area represents forebrain vesicle. No con-
sistent pattern of red fluorescence was detected among independent transgenic embryos. (G) Yolk sac from a second, independent trans-
genic specimen (single confocal slice view), with a strong fluorescence signal. (H) Yolk sac from a nontransgenic specimen without fluo-
rescent reporter activity.

moter, (3) the transcription factor TGIF from the CMV                  1999a, 1999b), silencing of the construct might have been
promoter, or (4) the red fluorescent protein HcRed from                 expected, but was not observed. Surprisingly, the F1CE2-
the CMV promoter. We used CMV/HcRed to control for                     F1P4 deletion construct carrying the presumed HNF4-
the presence of a very strong enhancer/promoter                        alpha site, showed significantly lower activation in
sequence in the transfected cells, and any sequestering of             response to HNF4-alpha than the construct carrying the
general transcription factors that might occur because of              intact F1CE2 enhancer element. The construct carrying
the CMV sequence. To test whether any observed effect                  the F1P4 promoter alone also responded to the presence
would be due to the function of a sequence-specific DNA                 of HNF4-alpha, although not nearly as strong as the con-
binding protein and not just due to the increased pres-                struct with the F1CE2 enhancer. Therefore, it is likely
ence of any DNA-binding protein, we used the transcrip-                that transcriptional activation via HNF4-alpha involves
tion factor TGIF, which is not related to the biologic con-            more than a single binding site on F1CE2. Taken to-
text of the experiment. All reporter activities were nor-              gether, these results suggest HNF4-alpha as a part of the
malized to the CMV/HcRed co-transfection to calculate                  regulatory mechanism that controls folate receptor 1 gene
fold-change as a response to presence of HNF4-alpha. In                expression specifically in the visceral endoderm and the
these experiments, we observed that presence of HNF4-                  yolk sac.
alpha lead to a robust and highly significant increase of
reporter activity from the F1CE2-F1P4 construct com-                                           DISCUSSION
pared to control (Fig. 6). The degree of increase was dif-
ferent for the two HNF4-alpha expression plasmids (60-                    In this study, we demonstrate that a DNA sequence
fold for CMV vs 20-fold for MT7). One reason may be                    located approximately 13 kb upstream of the P4 pro-
that the two plasmids express different splice variants of             moter of the human FOLR1 gene can act as a transcrip-
HNF4-alpha, which are thought to have slightly different               tional enhancer for FOLR1 gene expression in the visceral
transcriptional activity (Eeckhoute et al., 2003). More                endoderm and the yolk sac. Whereas the P4 promoter
likely though, the difference is due to the higher degree              displays activity in an in vitro model of visceral endo-
of expression of HNF4-alpha from the CMV promoter                      derm, it appears that neither the P4 nor the P1 promoter
plasmid compared with the MT7 promoter plasmid. As                     of the human or the murine Folr1 genes alone contain the
expected, cotransfection of TGIF as a control for                      necessary regulatory elements to drive expression of this
increased DNA binding protein content in the cells did                 gene properly. Addition of the evolutionary conserved
not affect the F1CE2-F1P4 construct in a significant way:               sequence F1CE2 to P4 reporter constructs confers
as TGIF is a transcriptional co-repressor (Wotton et al.,              increased transcriptional activity from the construct

                                                                                   Birth Defects Research (Part A) 85:303À313 (2009)
310                                                     SALBAUM ET AL.
                                                                   tory elements). Therefore, if reporter gene expression
                                                                   matches between different transgenic founders, it is a
                                                                   strong indication that the observed reporter gene expres-
                                                                   sion is due to a biologic function on the transgene
                                                                   sequence, and not due to the genomic integration site.
                                                                   The fact that we observed excellent congruency of re-
                                                                   porter expression at both developmental time points is a
                                                                   compelling argument that the F1CE2 sequence harbors
                                                                   transcriptional enhancer function. The full characteriza-
                                                                   tion of the F1CE2 enhancer (e.g., the developmental time
                                                                   course) will have to await the establishment of transgenic
                                                                   mouse lines.
                                                                      Our experiments show that the F1CE2 sequence has in-
                                                                   structive properties and is sufficient to drive expression
                                                                   in the visceral endoderm. However, we used only the P4
                                                                   promoter of FOLR1 in the pertinent experiments. We can-
                                                                   not rule out that the F1CE2 enhancer could also activate
                                                                   the P1 promoter of FOLR1. Preliminary experiments (not
                                                                   shown) using a heterologous promoter from the ICP4
Figure 6. HNF4alpha can activate the F1CE2 sequence from the       gene of herpes simplex virus indicate that the F1CE2
FOLR1 gene. Cotransfection experiments indicate that the con-      sequence can activate such a heterologous promoter at
struct carrying the entire F1CE2 enhancer sequence responds        least in F9 cells differentiated to visceral endoderm, and
very strongly to the presence of HNF4-alpha. F1P4-GL3, human
FOLR1 P4 promoter fused to a luciferase reporter; F1CE2-F1P4-
                                                                   thereby fulfill the classic definition of an enhancer. It
GL3, the F1P4-GL3 construct carrying the entire F1CE2 enhancer     therefore stands to reason that the P1 promoter of the
sequence; F1CE2-dPSM-F1P4-GL3, the F1P4-GL3 construct with         FOLR1 gene may also be activated by the F1CE2
a deletion version of the F1CE2 enhancer. A plasmid expression     sequence, although this remains to be proven experimen-
HcRed (instead of any transcription factor) from the CMV pro-      tally.
moter was used as control. Values were normalized to the aver-        Although our data demonstrate the biologic activity of
age of the control experiment to determine fold-changes.           F1CE2, our studies cannot address whether the F1CE2
Cotransfection of HNF4-alpha leads to a strong activation of the   sequence is solely responsible, or even necessary for vis-
reporter construct carrying the entire F1CE2 sequence, with only   ceral endoderm expression of FOLR1. In fact, the DNA
mild increases seen for the F1P4 promoter alone, or for the con-
struct with the deletion version of the F1CE2 sequence.
                                                                   sequence conservation at the FOLR1 gene locus would
                                                                   suggest that there may be other sequences in the vicinity
                                                                   of the FOLR1 gene that may have similar properties as
                                                                   F1CE2. Closer examination of the F1CE2 sequence
                                                                   revealed that F1CE2 is in fact a remnant of a folate recep-
in vitro, and allows tissue-specific expression congruent           tor gene, or a folate receptor pseudogene. No transcripts
with Folr1 gene expression in transgenic experiments               have been reported to arise from the human F1CE2
in vivo. These results are consistent with our interpreta-         sequence, but a close sequence relationship for three
tion that the F1CE2 sequence functions as an enhancer.             small subregions on F1CE2 to the last three coding exons
   In this context, it is important to note that in the ab-        of other folate receptor genes is readily recognizable (Fig.
sence of developmental expression data for the human               7). The F1CE2 sequence has a positional match in primate
FOLR1 gene, we are using the mouse Folr1 gene and its              genomes, as well as in the genomes of dog and horse: in
expression as a model and as guidance to evaluate the              these genomes, a sequence matching F1CE2 exists in a
activity of sequences from the human FOLR1 gene locus.             location upstream of the cognate folate receptor 1 gene.
The fact that the human DNA sequences used in this                 Interestingly, no such positional match exists between the
study were able to generate specific transcriptional
responses in our in vitro model of murine origin, as well
as in transgenic mouse experiments in vivo, would argue
that regulatory mechanisms are conserved between the
human and mouse version of the folate receptor 1 gene.
This would suggest that expression of the human gene
may occur in a manner similar to the mouse gene during
embryonic development.
   We used a strategy of analyzing the transgenic speci-
men directly; in this fashion, every reporter expression
signal arose from an independent transgenic event. Since
transgene DNA introduced by pronuclear injection typi-             Figure 7. Conservation of the F1CE2 sequence. Comparison of
cally integrates in a random manner, it is highly unlikely         the F1CE2 sequence to human and mouse genomes revealed the
                                                                   presence of three conserved regions (dark shading). The match
that two independent transgenic events occur at the same
                                                                   to the F1CE2 sequence itself in the human genome is not shown.
genomic integration site. Consequently, the genomic                Capitalized gene names are human genes, u-F1CE2 denotes a
neighborhood is unique for each transgenic event. The              sequence upstream of the F1CE2 sequence in the human genome
genomic neighborhood of a transgene can exert strong               that is also a remnant of a folate receptor gene. Percentage of
influences on a transgene expression (e.g., through meth-           sequence identity over a given nucleotide span is indicated. The
ylation patterns or through the presence of strong regula-         regions of 525bp and 177bp are not drawn to scale.

Birth Defects Research (Part A) 85:303À313 (2009)
                           TRANSCRIPTIONAL ENHANCER FOR FOLATE RECEPTOR 1                                                           311
human and mouse genomes. In the mouse, the only              mice, or the removal of potential HNF4-alpha binding
sequences with relationship to F1CE2 are in fact the         sites from the F1CE2 enhancer sequence. Given that there
sequences for the two folate receptor genes Folr1 and        are other conserved potential transcription factor binding
Folr2. In line with the hypothesis of conservation of        sites on the F1CE2 sequence, it is likely that F1CE2
expression and regulation, we propose that sequences         enhancer function requires other factors besides HNF4-
controlling the murine Folr1 gene may reside in either       alpha. Nevertheless, our results raise the possibility that
the Folr1 gene itself, or in the neighboring Folr2 gene.     folate transport is in fact a process controlled by HNF4-
Based on sequence similarity and on the presence of          alpha, and support the concept that HNF4-alpha func-
another potential HNF4-alpha binding site, it appears        tions to integrate the general process of nurturing the
that the Folr2 sequence is the more likely candidate for     embryo before the completion of the placenta and the
harboring enhancer function. It is therefore reasonable to   onset of hemotrophic nutrition.
assume that the human folate receptor gene locus on             This study provides a first insight into the mechanisms
chromosome 11 with it’s higher complexity of folate re-      that regulate FOLR1 gene expression during critical times
ceptor genes – besides FOLR1 and FOLR2, there is also        of embryonic development. Understanding the signals
the FOLR3 gene, as well as two FOLR gene remnants            that impinge on these mechanisms and their relationship
(one of which is F1CE2) – might harbor more than one         to maternal folate status will provide further insight into
sequence capable of driving FOLR1 expression in the vis-     the regulation of FOLR1 gene expression and folate trans-
ceral endoderm.                                              port in the embryo.
   It is well known that folate supplementation can
reduce the incidence of NTDs (Smithells et al., 1981). In
regard to the timing of neural tube closure, it is impor-                       ACKNOWLEDGMENTS
tant to note that the process of neurulation occurs at a       Part of this work was carried out at and funded by the
time when the chorioallantoic placenta has not been fully    Munroe-Meyer Institute at the University of Nebraska
established. At that time, the visceral endoderm has the     Medical Center. We wish to acknowledge technical help
function of supplying nutrients to the embryo. In the        by Andrew Wall, Ryan Taylor, and Don Harms, help
quest of understanding how folate supplementation can        with confocal microscopy by Dr. Bernd Fritzsch and
exert its benefits on the embryo, it appears that the func-   Heather Thomas, as well support by grants NIH
tion of the visceral endoderm is of high biologic signifi-    DE016315 (to RHF) and NIH DK063336 (to CK).
cance. In the mouse, the visceral endoderm mediates
histiotrophic nutrition of the embryo, and it stands to
reason that the presence of FOLR1 in cells of the visceral                              REFERENCES
endoderm is connected to folate uptake and transport to      Bailey LB. 2000. New standard for dietary folate intake in pregnant
the developing embryo just ahead and during the time of           women. Am J Clin Nutr 71(5 Suppl):1304S–1307S.
neurulation. Disturbance of this process may produce         Barber R, Shalat S, Hendricks K, et al. 2000. Investigation of folate path-
                                                                  way gene polymorphisms and the incidence of neural tube defects in
detrimental results for the embryo, and it appears that           a Texas hispanic population. Mol Genet Metab 70:45–52.
the regulatory mechanisms that ensure FOLR1 in the tis-      Beddington RS, Robertson EJ. 1998. Anterior patterning in mouse. Trends
sue that feeds the embryo during a crucial time play a            Genet 14:277–284.
very important role for proper development.                  Belaoussoff M, Farrington SM, Baron MH. 1998. Hematopoietic induction
                                                                  and respecification of A-P identity by visceral endoderm signaling in
   The role of the visceral endoderm in nutrition of the          the mouse embryo. Development 125:5009–5018.
embryo has been addressed from the viewpoint of tar-         Bielinska M, Narita N, Wilson DB. 1999. Distinct roles for visceral endo-
geted gene mutations. In particular, mouse embryos lack-          derm during embryonic mouse development. Int J Dev Biol 43:183–
ing the transcription factor HNF4-alpha fail to complete          205.
                                                             Boucher DM, Pedersen RA. 1996. Induction and differentiation of extra-
gastrulation due to malfunction of the visceral endoderm          embryonic mesoderm in the mouse. Reprod Fertil Dev 8:765–777.
(Chen et al., 1994; Duncan et al., 1994). In fact, it has    Braunhut SJ, D’Amore PA, Gudas LJ. 1992. The location and expression
been shown that HNF4-alpha controls the expression of             of fibroblast growth factor (FGF) in F9 visceral and parietal embry-
several genes that are important for the transport of             onic cells after retinoic acid-induced differentiation. Differentiation
nutrients. In HNF4-alpha-mutant embryos, genes for nu-       Brent RL, Beckman DA, Jensen M, Koszalka TR. 1990. Experimental yolk
trient transport or metabolism, such as apolipoproteins,          sac dysfunction as a model for studying nutritional disturbances in
glucose transporter 2, transferrin, and cytoplasmic reti-         the embryo during early organogenesis. Teratology 41:405–413.
noic acid binding proteins were downregulated in the         Burton GJ, Hempstock J, Jauniaux E. 2001. Nutrition of the human fetus
                                                                  during the first trimester–a review. Placenta 22 Suppl A:S70–77.
visceral endoderm, and it is thought that these embryos      Chen WS, Manova K, Weinstein DC, Duncan SA, Plump AS, Prezioso
die from starvation (Stoffel and Duncan, 1997). These             VR, Bachvarova RF, Darnell JE, Jr. 1994. Disruption of the HNF-4
findings suggest that HNF4-alpha could act as a master             gene, expressed in visceral endoderm, leads to cell death in embry-
control gene for nutrition of the embryo. Therefore, it           onic ectoderm and impaired gastrulation of mouse embryos. Genes
                                                                  Dev 8:2466–2477.
was interesting to find potential binding sites for HNF4-     Copp AJ. 1995. Death before birth: clues from gene knockouts and muta-
alpha on the F1CE2 enhancer sequence of the FOLR1                 tions. Trends Genet 11:87–93.
gene. As our experiments clearly demonstrate, the F1CE2      Copp AJ. 2005. Neurulation in the cranial region–normal and abnormal.
enhancer is highly responsive to the presence of HNF4-            J Anat 207:623–635.
                                                             Copp AJ, Brook FA, Roberts HJ. 1988. A cell-type-specific abnormality of
alpha, suggesting that FOLR1 expression in the visceral           cell proliferation in mutant (curly tail) mouse embryos developing
endoderm is driven by HNF4-alpha. The deletion analy-             spinal neural tube defects. Development 104:285–295.
sis further suggests that more than one HNF4-alpha site      Courtemanche C, Elson-Schwab I, Mashiyama ST, et al. 2004a. Folate defi-
may be involved in the function of the F1CE2 enhancer.            ciency inhibits the proliferation of primary human CD81 T lympho-
                                                                  cytes in vitro. J Immunol 173:3186–3192.
At this time, it is not known whether FOLR1 expression       Courtemanche C, Huang AC, Elson-Schwab I, et al. 2004b. Folate defi-
is strictly dependent on HNF4-alpha; such an analysis             ciency and ionizing radiation cause DNA breaks in primary human
would require either the study of HNF4-alpha-deficient             lymphocytes: a comparison. FASEB J 18:209–211.

                                                                          Birth Defects Research (Part A) 85:303À313 (2009)
312                                                                SALBAUM ET AL.
Cross JC, Werb Z, Fisher SJ. 1994. Implantation and the placenta: key          Page ST, Owen WC, Price K, Elwood PC. 1993. Expression of the human
     pieces of the development puzzle. Science 266:1508–1518.                       placental folate receptor transcript is regulated in human tissues. Or-
D’Anci KE, Rosenberg IH. 2004. Folate and brain function in the elderly.            ganization and full nucleotide sequence of the gene. J Mol Biol
     Curr Opin Clin Nutr Metab Care 7:659–664.                                      229:1175–1183.
Dong JM, Li F, Chiu JF. 1990. Induction of F9 cell differentiation by tran-    Piedrahita JA, Oetama B, Bennett GD, et al. 1999. Mice lacking the folic
     sient exposure to retinoic acid. Biochem Biophys Res Commun                    acid-binding protein Folbp1 are defective in early embryonic devel-
     170:147–152.                                                                   opment. Nat Genet 23:228–232.
Doucette MM, Stevens VL. 2001. Folate receptor function is regulated in        Prinz-Langenohl R, Fohr I, Pietrzik K. 2001. Beneficial role for folate in
     response to different cellular growth rates in cultured mammalian              the prevention of colorectal and breast cancer. Eur J Nutr 40:98–105.
     cells. J Nutr 131:2819–2825.                                              Qiu A, Jansen M, Sakaris A, et al. 2006. Identification of an intestinal fo-
Duncan SA, Manova K, Chen WS, Hoodless P, Weinstein DC, Bachvarova                  late transporter and the molecular basis for hereditary folate malab-
     RF, Darnell JE, Jr. 1994. Expression of transcription factor HNF-4 in          sorption. Cell 127:917–928.
     the extraembryonic endoderm, gut, and nephrogenic tissue of the           Qiu A, Min SH, Jansen M, et al. 2007. Rodent intestinal folate transporters
     developing mouse embryo: HNF-4 is a marker for primary endoderm                (SLC46A1): secondary structure, functional properties, and response to
     in the implanting blastocyst. Proc Natl Acad Sci U S A 91:7598–7602.           dietary folate restriction. Am J Physiol Cell Physiol 293:C1669–1678.
Duncan SA, Nagy A, Chan W. 1997. Murine gastrulation requires HNF-4            Reddy JA, Haneline LS, Srour EF, et al. 1999. Expression and functional
     regulated gene expression in the visceral endoderm: tetraploid rescue          characterization of the beta-isoform of the folate receptor on CD34(1)
     of Hnf-4(-/-) embryos. Development 124:279–287.                                cells. Blood 93:3940–3948.
Eeckhoute J, Moerman E, Bouckenooghe T, et al. 2003. Hepatocyte nuclear        Reece EA, Eriksson UJ. 1996. The pathogenesis of diabetes-associated con-
     factor 4 alpha isoforms originated from the P1 promoter are                    genital malformations. Obstet Gynecol Clin North Am 23:29–45.
     expressed in human pancreatic beta-cells and exhibit stronger tran-       Reece EA, Homko CJ, Wu YK, Wiznitzer A. 1993. Metabolic fuel mixtures
     scriptional potentials than P2 promoter-driven isoforms. Endocrinol-           and diabetic embryopathy. Clin Perinatol 20:517–532.
     ogy 144:1686–1694.                                                        Reece EA, Ji I, Wu YK, Zhao Z. 2006. Characterization of differential gene
Elwood PC. 1989. Molecular cloning and characterization of the human                expression profiles in diabetic embryopathy using DNA microarray
     folate-binding protein cDNA from placenta and malignant tissue cul-            analysis. Am J Obstet Gynecol 195:1075–1080.
     ture (KB) cells. J Biol Chem 264:14893–14901.                             Ross JF, Chaudhuri PK, Ratnam M. 1994. Differential regulation of folate recep-
Elwood PC, Nachmanoff K, Saikawa Y, et al. 1997. The divergent 50 ter-              tor isoforms in normal and malignant tissues in vivo and in established
     mini of the alpha human folate receptor (hFR) mRNAs originate                  cell lines. Physiologic and clinical implications. Cancer 73:2432–2443.
     from two tissue-specific promoters and alternative splicing: character-    Ross JF, Wang H, Behm FG, et al. 1999. Folate receptor type beta is a neu-
     ization of the alpha hFR gene structure. Biochemistry 36:1467–1478.            trophilic lineage marker and is differentially expressed in myeloid
Galmozzi E, Tomassetti A, Sforzini S, et al. 2001. Exon 3 of the alpha fo-          leukemia. Cancer 85:348–357.
     late receptor gene contains a 50 splice site which confers enhanced       Ryan BM, Weir DG. 2001. Relevance of folate metabolism in the pathoge-
     ovarian carcinoma specific expression. FEBS Lett 502:31–34.                     nesis of colorectal cancer. J Lab Clin Med 138:164–176.
Gelineau-van Waes J, Finnell RH. 2001. Genetics of neural tube defects.        Sadasivan E, Cedeno MM, Rothenberg SP. 1994. Characterization of the
     Semin Pediatr Neurol 8:160–164.                                                gene encoding a folate-binding protein expressed in human placenta.
Hogan BL, Costantini F, Lacy E. 1996. Manipulating the mouse embryo: a              Identification of promoter activity in a G-rich SP1 site linked with the
     laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor                  tandemly repeated GGAAG motif for the ets encoded GA-binding
     Laboratory Press.                                                              protein. J Biol Chem 269:4725–4735.
Jiang G, Nepomuceno L, Hopkins K, Sladek FM. 1995. Exclusive homodi-           Said HM, Nguyen TT, Dyer DL, et al. 1996. Intestinal folate transport:
     merization of the orphan receptor hepatocyte nuclear factor 4 defines           identification of a cDNA involved in folate transport and the func-
     a new subclass of nuclear receptors. Mol Cell Biol 15:5131–5143.               tional expression and distribution of its mRNA. Biochem Biophys
Kappen C, Mello MA, Finnell RH, Salbaum JM. 2004. Folate modulates                  Acta 1281:164–172.
     Hox gene-controlled skeletal phenotypes. Genesis 39:155–166.              Saikawa Y, Price K, Hance KW, et al. 1995. Structural and functional anal-
Kelley KM, Rowan BG, Ratnam M. 2003. Modulation of the folate recep-                ysis of the human KB cell folate receptor gene P4 promoter: coopera-
     tor alpha gene by the estrogen receptor: mechanism and implications            tion of three clustered Sp1-binding sites with initiator region for basal
     in tumor targeting. Cancer Res 63:2820–2828.                                   promoter activity. Biochemistry 34:9951–9961.
Kim JM, Stewart R, Kim SW, et al. 2008. Changes in folate, vitamin B12         Saitsu H, Ishibashi M, Nakano H, Shiota K. 2003. Spatial and temporal
     and homocysteine associated with incident dementia. J Neurol Neu-              expression of folate-binding protein 1 (Fbp1) is closely associated
     rosurg Psychiatry 79:864–868.                                                  with anterior neural tube closure in mice. Dev Dyn 226:112–117.
Lacey SW, Sanders JM, Rothberg KG, et al. 1989. Complementary DNA              Salbaum JM. 1998. Punc, a novel mouse gene of the immunoglobulin
     for the folate binding protein correctly predicts anchoring to the             superfamily, is expressed predominantly in the developing nervous
     membrane by glycosyl-phosphatidylinositol. J Clin Invest 84:715–720.           system. Mech Dev 71:201–204.
Ladipo OA. 2000. Nutrition in pregnancy: mineral and vitamin supple-           Shaw GM, Schaffer D, Velie EM, et al. 1995. Periconceptional vitamin use,
     ments. Am J Clin Nutr 72(1 Suppl):280S–290S.                                   dietary folate, and the occurrence of neural tube defects. Epidemiol-
Locksmith GJ, Duff P. 1998. Preventing neural tube defects: the impor-              ogy 6:219–226.
     tance of periconceptional folic acid supplements. Obstet Gynecol          Shen F, Ross JF, Wang X, Ratnam M. 1994. Identification of a novel folate
     91:1027–1034.                                                                  receptor, a truncated receptor, and receptor type beta in hematopoi-
Loots GG, Ovcharenko I. 2004. rVISTA 2.0: evolutionary analysis of tran-            etic cells: cDNA cloning, expression, immunoreactivity, and tissue
     scription factor binding sites. Nucleic Acids Res 32(Web Server                specificity. Biochemistry 33:1209–1215.
     issue):W217–221.                                                          Singh M. 2004. Role of micronutrients for physical growth and mental de-
Loots GG, Ovcharenko I, Pachter L, et al. 2002. rVista for comparative              velopment. Indian J Pediatr 71:59–62.
     sequence-based discovery of functional transcription factor binding       Smith SB, Kekuda R, Gu X, et al. 1999. Expression of folate receptor alpha
     sites. Genome Res 12:832–839.                                                  in the mammalian retinol pigmented epithelium and retina. Invest
Lukaski HC. 2004. Vitamin and mineral status: effects on physical per-              Ophthalmol Vis Sci 40:840–848.
     formance. Nutrition 20:632–644.                                           Smithells RW, Ankers C, Carver ME, et al. 1977. Maternal nutrition in
Maddox DM, Manlapat A, Roon P, et al. 2003. Reduced-folate carrier                  early pregnancy. Br J Nutr 38:497–506.
     (RFC) is expressed in placenta and yolk sac, as well as in cells of the   Smithells RW, Sheppard S, Schorah CJ. 1976. Vitamin deficiencies and
     developing forebrain, hindbrain, neural tube, craniofacial region, eye,        neural tube defects. Arch Dis Child 51:944–950.
     limb buds and heart. BMC Dev Biol 3:6.                                    Smithells RW, Sheppard S, Schorah CJ, et al. 1981. Apparent prevention
Milunsky A, Jick H, Jick SS, et al. 1989. Multivitamin/folic acid supple-           of neural tube defects by periconceptional vitamin supplementation.
     mentation in early pregnancy reduces the prevalence of neural tube             Arch Dis Child 56:911–918.
     defects. JAMA 262:2847–2852.                                              Spiegelstein O, Eudy JD, Finnell RH. 2000. Identification of two putative
Molloy AM, Scott JM. 2001. Folates and prevention of disease. Public                novel folate receptor genes in humans and mouse. Gene 258:117–125.
     Health Nutr 4:601–609.                                                    Spiegelstein O, Mitchell LE, Merriweather MY, et al. 2004. Embryonic de-
Moscow JA, Gong M, He R, et al. 1995. Isolation of a gene encoding a                velopment of folate binding protein-1 (Folbp1) knockout mice: Effects
     human reduced folate carrier (RFC1) and analysis of its expression in          of the chemical form, dose, and timing of maternal folate supplemen-
     transport-deficient, methotrexate-resistant human breast cancer cells.          tation. Dev Dyn 231:221–231.
     Cancer Res 55:3790–3794.                                                  Stoffel M, Duncan SA. 1997. The maturity-onset diabetes of the young
Nilsson TK, Borjel AK. 2004. Novel insertion and deletion mutations in              (MODY1) transcription factor HNF4alpha regulates expression of
     the 50 -UTR of the folate receptor-alpha gene: an additional contribu-         genes required for glucose transport and metabolism. Proc Natl Acad
     tor to hyperhomocysteinemia? Clin Biochem 37:224–229.                          Sci U S A 94:13209–13214.

Birth Defects Research (Part A) 85:303À313 (2009)
                                     TRANSCRIPTIONAL ENHANCER FOR FOLATE RECEPTOR 1                                                                    313
Tam PP, Behringer RR. 1997. Mouse gastrulation: the formation of a                 Wang Y, Zhao R, Russell RG, Goldman ID. 2001. Localization of the mu-
    mammalian body plan. Mech Dev 68:3–25.                                             rine reduced folate carrier as assessed by immunohistochemical anal-
Thomas P, Beddington R. 1996. Anterior primitive endoderm may be re-                   ysis. Biochim Biophys Acta 1513:49–54.
    sponsible for patterning the anterior neural plate in the mouse                Wong SC, Proefke SA, Bhushan A, Matherly LH. 1995. Isolation of human
    embryo. Curr Biol 6:1487–1496.                                                     cDNAs that restore methotrexate sensitivity and reduced folate car-
Tomassetti A, Mangiarotti F, Mazzi M, et al. 2003. The variant hepatocyte              rier activity in methotrexate transport-defective Chinese hamster
    nuclear factor 1 activates the P1 promoter of the human alpha-folate               ovary cells. J Biol Chem 270:17468–17475.
    receptor gene in ovarian carcinoma. Cancer Res 63:696–704.                     Wotton D, Lo RS, Lee S, Massague J. 1999a. A Smad transcriptional core-
Trippett TM, Bertino JR. 1999. Therapeutic strategies targeting proteins that          pressor. Cell 97:29–39.
    regulate folate and reduced folate transport. J Chemother 11:3–10.             Wotton D, Lo RS, Swaby LA, Massague J. 1999b. Multiple modes of
van Straaten HW, Hekking JW, Consten C, Copp AJ. 1993. Intrinsic and extrin-           repression by the Smad transcriptional corepressor TGIF. J Biol Chem
    sic factors in the mechanism of neurulation: effect of curvature of the body       274:37105–37110.
    axis on closure of the posterior neuropore. Development 117:1163–1172.         Wu M, Fan J, Gunning W, Ratnam M. 1997. Clustering of GPI-anchored
Wald N, Sneddon J, Densem J, et al. 1991. Prevention of neural tube                    folate receptor independent of both cross-linking and association
    defects: results of the Medical Research Council Vitamin Study. MRC                with caveolin. J Membr Biol 159:137–147.
    Vitamin Study Research Group. Lancet 338:131–137.                              Yamaguchi T, Hirota K, Nagahama K, et al. 2007. Control of immune
Walker LS. 2007. Regulatory T cells: Folate receptor 4: a new handle on                responses by antigen-specific regulatory T cells expressing the folate
    regulation and memory? Immunol Cell Biol 85:506–507.                               receptor. Immunity 27:145–159.
Wang X, Jansen G, Fan J, et al. 1996. Variant GPI structure in relation to         Yates JR, Ferguson-Smith MA, Shenkin A, et al. 1987. Is disordered folate
    membrane-associated functions of a murine folate receptor. Biochem-                metabolism the basis for the genetic predisposition to neural tube
    istry 35:16305–16312.                                                              defects? Clin Genet 31(5):279–287.

                                                                                                Birth Defects Research (Part A) 85:303À313 (2009)

Shared By: